The retinoblastoma (Rb) protein is a key cell-cycle regulator disrupted in many cancers, and thus a molecular picture of Rb function is important for understanding mechanisms of tumorigenesis. Rb binds members of the E2F transcription factor family and thereby inhibits the expression of genes required for cell-cycle progression. Multiple phosphorylations of Rb dissociate Rb-E2F complexes, and this process is regulated by Cyclin-dependent kinases (Cdks) and protein phosphatase 1 (PP1). The objective of this proposal is to understand in molecular detail how phosphorylation disrupts Rb-E2F binding and how Cdks and PP1 phosphorylate and dephosphorylate Rb. A number of biophysical and structural techniques including x-ray crystallography, nuclear magnetic resonance, mass spectrometry, and calorimetry, will be applied to characterize the protein interactions of Rb with E2F, Cdks and PP1. Tumor cells invariably have defects in the biochemical mechanisms that regulate cell growth and division, so understanding how these processes work is vital to understanding cancer. This proposal aims to develop a molecular picture of how a cell-cycle regulator known as the retinoblastoma protein functions.